Atlas of Antinuclear Antibodies

Atlas of Antinuclear Antibodies
　　　Ribosome
　　　PCNA
　　　Lysosome
　　Mitochondria
　　　Golgi complex
The autoantibody testing by indirect immunofluorescence (IIF) has fulfilled the very important roles for the diagnosis and
treatment of the patients with autoimmune disorders since its first report by Friou in 1957.
The technique has many advantages for detecting and screening both nuclear and cytoplasmic antibodies in general and
categorizing them by their staining patterns. Since the recognized antigens of the ANA (antinuclear antibodies) comprise many
nuclear components or proteins and sometimes it is difficult to distinguish them by their staining patterns, these studies have
contributed much to the elucidation and understanding of the autoantibodies in connective tissue diseases.
* Photos of cell staining in this photo book were taken by using FLUORO HEPANA TEST (code No. 4210, 4220). Specific staining parts were
detected as fluorescent staining (green) and non specific staining parts would be appeared as red because of Evans Blue pigment. Depends on
cases, when both parts were over lapped, it would look yellow.
Structure and function of eukaryotic cell and outlines of cell cycle
One of the major advantages of detecting and screening autoantibodies by IIF is to find out the localization of recognized cellular
antigens in various cell cycles by their staining patterns. It becomes very important to realize the structure of the cell related to
the function and localization of the antigens in the cell.
■ Structure and function of cell endoplasmic reticulum nucleus nuclear membrane

nucleolus mitochondria
General structure of the eukaryotic cell as HEp-2 cell ribosome
lysosome
is shown in Fig. 1. Inside the cell, various organelles
including the nucleus, endoplasmic reticulum, golgi
apparatus, mitochondria, peroxisomes and lysosomes
are recognized. The cell surface is covered by plasma
membrane, and the space between the plasma cell membrane
membrane and organelles are occupied by insoluble

cytoplasm
cytoskelton and soluble cytoplasm.
centriole cytoskeleton Golgi apparatus
Fig. 1 HEp-2 cell

Nucleus
The nucleus is covered by bi-layer nuclear membrane. The outer nuclear membrane is connected with endoplasmic reticulum
and transport of various substances between the nucleus and cytoplasm occurs through nuclear pore complexes. The inner
nuclear membrane is connected with lamina. In interphase cells, chromosomes are dispersed into chromatins throughout the
nucleoplasm. The chromatins are composed of DNA, histones, and other non-histone proteins. In the granular components
of nucleus, precursor mRNA (heterogeneous nuclear RNA; hnRNA) and small RNA-protein complexes (small nuclear
ribonucleoproteins; snRNP) for splicing of hnRNA can be found.
In the nucleolus, ribosomal RNA precursors are transcribed and after their processing and assembly, ribosomes are transported
to the cytoplasm.
Cytoplasm
Endoplasmic reticulum (ER)

ER are divided into rough surface RER which binds ribosomes on its surface and smooth surface SER. RER is the main site of
protein synthesis in the cell and the SER is found abundantly in cells like Leydig cells in the testis which synthesize and secrete
steroid hormones. Oxidoreductase systems of NADH and NADPH in the SER membrane are concerned with unsaturation and
ω-oxidation of fatty acids and detoxication of xenobiotic substances.
Golgi apparatus
Golgi apparatus is composed of stacks of cisterna and divided into cis, medial and trans regions. It is mainly concerned with the
modification (addition of sugar chains) and sorting of the synthesized proteins in RER for secretion and transport in cells.
Mitochondria
Mitochondria are oval in structure. Respiratory chain and ATP synthetase complex for ATP production are in its inner membrane.
Enzymes for TCA cycle, fatty acid oxidation and amino acid metabolism, and own protein synthesis system with mitochondrial
DNA are in its matrix.
Lysosome
Lysosome is an organelle with monolayer membrane, and it is mainly concerned with the hydrolysis of waste products and toxic
substances (proteins, mucopolysaccharides, glycolipids and nucleic acids) by their respective enzymes localized in the matrix
where low pH is maintained by the action of H+-ATPase.
2
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Table 1 Antigens recognized by autoantibodies in connective tissue diseases
antinuclear antibodies localization recognized antigens relevant diseases
anti-ssDNA antibody chromatins single stranded DNA (ssDNA) SLE etc.

anti-dsDNA antibody double-stranded DNA (dsDNA) SLE
anti-polyADP ribose antibody poly ADP ribose SLE
anti-Scl-70 antibody DNA topoisomerase I SSc
anti-centromere antibody centromere CREST
anti-histone antibody histone Drug induced lupus
anti-U1RNP antibody nucleus U1RNP MCTD

anti-Sm antibody U1, U2, U4/U6, U5 RNP SLE
anti-U2RNP antibody U2RNP Overlap syndrome
anti-SS-A/Ro antibody hY1-hY5 RNP SS etc.
anti-SS-B/La antibody RNA polymerase III transcription SS
anti-PCNA antibody DNA polymerase δ auxiliary factor SLE
anti-Ku antibody DNA-dependent protein kinase activation factor Overlap syndrome
anti-U3RNP antibody nucleolus U3RNP (Fibrillarin) SSc

anti-7-2(Th)RNP antibody RNaseP, RNaseMRP SSc
anti-RNA polymerase I antibody RNA polymerase I SSc
anti-PM-Scl antibody 11 proteins complexproteins complex (20-110 kD) Overlap syndrome
anti-NOR-90 antibody nucleolus NOR (hUBF)* SSc

anticytoplasmic antibodies localization recognized antigens relevant diseases
anti-Jo-1 antibody cytoplasm histidyl-tRNA synthetase PM/DM

anti-ribosomal antibody 60S ribosomal subunit SLE
anti-mitochondrial antibody mitochondria PBC
anti-SS-A/Ro antibody hY1-hY5 RNP SS etc.
others (anti actin antibody etc.)
*human Upstream Binding Factor
SLE: systemic lupus erythematosus, SSc: systemic sclerosis, MCTD: mixed connective tissue disease, SS: Sjögren's syndrome, PM/DM:
polymyositis/dermatomyositis, RA: rheumatoid arthritis, PBC: primary biliary cirrhosis, AIH: autoimmune hepatitis
Cell cycle
The cycle of the cell can be divided into interphase and mitosis (M phase). The interphase is subdivided into 3 successive
periods: G1(Gap 1), S(Synthesis), and G2(Gap 2) phases. The dispersed chromatin network of the nucleoplasm in the
interphase begins to condense in M phase, assembling into pairs of chromatids linked to each other at their centromeres.
Mitosis is divided into prophase, prometaphase, metaphase, anaphase and telophase.
*Staining patterns of anti-centromere antibody in photo.
3
4
1) Prophase 2) Prometaphase 3) Metaphase
plasma membrane
spindle pole
cytoplasm
nuclear membrane
nucleolus
kinetochore
separated centrosomes
become spindle poles. a part of plasma membrane
In prophase, cytoplasmic microtubles disrupt kinetochore spindle pole Chromatids (chromosomes) move and
microtubules
and the mitotic spindles begin to form around In prometaphase, the nuclear membrane arrange at the equator (metaphase
the nucleus and terminate at the polar entrioles begins to disrupt and kinetochore structures plate) of the spindle apparatus by the
plasma membrane to form bipolar structure. form around each centromere of sister action of kinetochore microtubules.
cytoplasm chromatids and attach to kinetochore 4) Anaphase
nuclear membrane microtubules of the mitotic spindle
nucleolus
apparatus. The spindle poles are also
centriole connected by the polar microtubules.
G2 kinetochore
Mitosis (M phase) microtubules
bocome shorten.
Interphase M
Adhesion cells like HEp-2 cells extend the S mitosis
cy toplasm and stick to the sur face of the
slide s. In G1 phase , DNA synthe sis is 6) Cytokinesis The chromosomes move separately to
prepared and histones start to synthesize. Cell Cycle each pole of the spindle.
DNA polymerase duplicates DNA during S
phase. In G2 phase, before cell division, DNA
duplication is checked for completeness. In
interphase 5) Telophase
interphase, the chromatins are dispersed all
over the nucleus.
G1
midbody
microtubule arrangement is reformed,

with the centrosomes at center.
After mitosis, cytokinesis occurs by the formation of

the contractile ring composed of actin filaments around Chromosomes reach opposite poles and microtubules
the middle of the cell, between daughter nuclei. The d i s a p p e a r. N u c l e a r m e m b r a n e r e f o r m s a r o u n d
cytoplasm in the cleavage furrow becomes midbody chromosomes which are then de condense d in the
which is progressively constricted and disappears as nucleus and nucleoli reappear.
G0
the daughter cells separate.
Fluorescence patterns of autoantibodies
■ Typical patterns of ANA using HEp-2 cells

Rodent tissue sections which were used as substrates during early periods of ANA testing have been replaced by HEp-2 cells
(immortalized cells orginated from human laryngeal carcinoma) grown as a monolayer on the slide. The HEp-2 cells have much
larger structures and higher cell division rates, so the detailed nuclear and cytoplasmic staining patterns can be recognized in
the cell populations of different cell cycles. It is noted that HEp-2 cells exhibit more than 30 different patterns, some are antigen-
specific but some are similar with different specificities, and the mixed patterns by different autoantibodies in patients' sera are
frequently observed. etailed nuclear and cytoplasmic staining patterns can be recognized in the cell populations of different cell
cycles. It is noted that HEp-2 cells exhibit more than 30 different patterns, some are antigen-specific but some are similar with
different specificities, and the mixed patterns by different autoantibodies in patients' sera are frequently observed.
Negative
No significant nuclear and cytoplasmic staining. Depending on the

sensitivity of the fluorescence microscope, faint fluorescence can
be observed, which is considered negative.
photo 1
In case of positive, staining patterns are interpreted.
Homogeneous nuclear staining
Uniform diffuse fluorescence staining of the entire nucleus in

interphase cells. The chromosomal regions in mitotic cells give
intense fluorescence.
photo 2
HEPASERA-I**
** HEPASERA-1 is MBL's ANA reference sera (Homogeneous pattern, Speckled pattern, Nucleolar pattern, Discrete speckled pattern).
Fluorescence patterns of autoantibodies 5

Peripheral nuclear staining
Uniform diffuse nuclear staining similar to homogeneous staining,

with greater intensity at its outer rim. The chromosomal region in
mitotic cells has uniform staining with greater intensity at its outer
rim. Most of the sera showing this pattern give homogeneous
staining when sera are diluted.
photo 3
Speckled nuclear staining
Fine or coarse speckles are distributed in the entire nucleus of

interphase cells. The chromosomal region shows no flurorescence
in most of the mitotic cells.
photo 4
HEPASERA-I**
Centromere (Discrete speckled) staining
40 to 80 discrete speckles are stained in the nucleus of interphase

cells. Speckles aligned in the chromosomal region of mitotic cells
can be seen.
photo 5
HEPASERA-I**
6 Fluorescence patterns of autoantibodies

Nucleolar staining
Homogeneous or speckled (clumpy) staining of the nucleolus of

interphase cells are seen by various nucleolar autoantibodies.
photo 6
HEPASERA-I**
Other staining patterns and antigens,
Cell cycle-dependent pleomorphic proliferating cell nuclear antigen (PCNA)

staining
Nuclear dots staining minute nuclear antigens; p80 coilin, Sp100 etc.
Nuclear membranous staining lamin, nuclear pore complex
Cytoplasmic staining cytoplasmic antigens; enzymes such as aminoacyl-tRNA synthetases, organelles

such as mitochondria and ribosome, cytoskelton, mitotic spindle apparatus
** HEPASERA-1 is MBL's ANA reference sera (Homogeneous pattern, Speckled pattern, Nucleolar pattern, Discrete speckled pattern).
Fluorescence patterns of autoantibodies 7

■ Mixed patterns
Mixed staining patterns are observed in a patient's serum containing more than one autoantibody. Since it is sometimes
difficult to distinguish different autoantibodies, the patterns can often be confirmed by titrating out the serum for its quantitative
evaluation. It is also useful to observe the staining of the chromosomal region and surrounding cytoplasm in mitotic cells. The
following photos show examples of mixed patterns.
indicate staining of the homogeneous, speckled and the mixed patterns respectively
photo 7 photo 8
Speckled nuclear staining Homogeneous nuclear staining
photo 9
Speckled nuclear staining and Homogeneous
nuclear staining mixed
The serum sample with the speckled pattern (photo 7) was mixed with another serum with homogeneous pattern (photo 8) at
1:1 ratio. The mixed sample shows the speckled pattern in interphase cells combined with chromosomal staining of mitotic cells.
(photo 9) The staining pattern is suggestive of coexistence of the speckled and the homogeneous patterns.
8 Mixed patterns
show another example of the mixed pattern
photo 10 photo 11
Anti-centromere antibody Anti-mitochondrial antibody
photo 12
The characteristic staining of nucleus and cytoplasm
suggestive of the coexistence of both antibodies
A serum sample with the anti-centromere antibody (photo 10) and the anti-mitochondrial antibody (photo 11) gives characteristic
staining of nucleus and cytoplasm suggestive of the coexistence of both antibodies (photo 12).
Mixed patterns 9
Fluorescence patterns of nuclear autoantigens
■ Autoantibodies recognizing chromatin related antigens
chromatin fiber
DNA
octameric histone core
(two each of H2A,H2B,
H3,H4 proteins)
DNA
chromosome "beads-on-a-string" form of chromatin

nucleosome, the fundamental structural unit of chromatin,
consists of DNA wound around a core particle of histone proteins
Chromatins are composed of dsDNA and its binding proteins (histones and non-histone proteins). 146 base pairs of dsDNA wind
around histone core (histone octamer composed of 2 molecules each of H2A, H2B, H3 and H4 subunits) to make a nucleosomal
unit which connect to each other through a DNA linker with histone H1 subunit. In interphase cells, chromatins are dispersed in
the nucleoplasm and condensed to form chromatids (chromosomes) in mitotic cells.
Anti-dsDNA antibody
photo13 photo14 　　AF/CDC-1*

Peripheral staining Homogeneous staining
10 Autoantibodies recognizing chromatin related antigens

photo15
kinetoplast of Clithidia luciliae is stained.
Pattern Peripheral and homogeneous staining in interphase cells with strong chromosomal staining in mitotic
cells. At lower serum dilutions (x 20-x 40), the antibodies tend to give peripheral patterns and often show
homogeneous staining at higher serum dilutions. Peripheral patterns are more frequently found when
rodent tissue sections are used as substrates.
Antigen Double stranded DNA(dsDNA). Anti-ssDNA antibodies do not give fluorescence.
Other analytical IIF, ELISA, RIA, CLEIA

method
Clinical The antibody is found flequently and specifically in SLE patients with active renal disease. The presence
significance of Anti-dsDNA is a major criteria for the classification of SLE.
References 5, 6
Anti-DNA histone complex antibody (anti-DNP antibody / anti-nucleosomal antibody)
Pattern Homogeneous nuclear staining in

interphase cells and intense fluorescence
in the chromosomal region in mitotic cells.
Antigen Histone-DNA complex with the antigenic

epitope in DNA-histone binding sites.
Other ELISA
analytical
method
Clinical SLE, lupus nephritis

significance
photo16 References 7, 8
* Reference serum from Centers for Disease Control

Autoantibodies recognizing chromatin related antigens 11

Anti-histone antibody
Pattern Similar to the former antibody
Antigen Histone H1, H2A, H2B, H3, H4, H2A-H2B complex (major antigen in IIF method) and H3-H4 complex.
Other analytical ELISA, WB

method
Clinical SLE (antibodies to H1 and H2A), drug-induced lupus (antibodies to H2A-H2B complex)
significance
References 9
Anti-Ku antibody
Pattern Speckled (patchy) nuclear staining (sometimes

described as homogeneous staining) with no
chromosomal staining in mitotic cells.
Monoclonal antibodies to the 70kD subunit
give similar patterns.
Antigen DNA binding 80kD/70kD heterodimer protein

(DNA-dependent protein kinase activator).
Antibodies targeting either subunit or the
complex are reported.
Other DID, Immunoprecipitation

photo17 analytical
method
Clinical SLE, SSc (USA) / polymyositis/scleroderma

significance overlap syndrome (Japan)
References 1, 4, 10, 11
Anti-Scl-70 antibody (anti-Topoisomerase I antibody)
Pattern Homogeneous (dense fine speckled to

homogeneous) staining with chromosomal
staining in interphase cells.
Nucleolar staining is often observed.
Antigen DNA Topoisomerase I
Other DID, ELISA, WB, CLEIA

analytical
method
Clinical Antibodies to topoisomerase I are generally

significance found in systemic sclerosis with high
photo 18 sensitivity and their presence constitutes one
AF/CDC-9* of the diagnostic criteria of the disease.
References 12
12 Autoantibodies recognizing chromatin related antigens

Anti-centromere antibody
photo 19 photo 20
AF/CDC-8* 2 fluorescent dots are visible for each chromosomal pair.
(hypotonic spread preparation of mitotic chromosomes)
Pattern Centromere or discrete speckled staining. 40-80 fine speckles are stained in the nucleus of interphase cells.
The speckles are characteristically aligned at the chromosomal region in mitotic cells. Paired centromeric
fluorescence staining is observed in the chromosomal spread preparations.
Antigen Centromere proteins; CENP-B (80kD), CENP-A (17kD), CENP-C (140kD)
Other ELISA, WB, CLEIA

analytical
method
Clinical These antibodies are consistently found in patients with the limited form of SSc and their presence constitutes
significance a diagnostic criteria of SSc. These antibodies are also found in PBC.
References 13, 14
pairing domain
kinetochore
fibrous corona
CENP-E,
dynein
outer plate centromere

CENP-F heterochromatin
middle zone CENP-B 80kD
3F3/2 antigen inner plate
CENP-C 140kD
The constricted region where two sister chromatids join along is the centromere. CENP is a specific protein in this region.
Microtubules of the spindle apparatus attach to kinetochore when the chromatid separation occurs.

Autoantibodies recognizing chromatin related antigens 13

■ Autoantibodies recognizing non-chromatin related proteins
DNA
RNA polymerase II
precursor mRNA
(hnRNA) introns introns
U2RNP
splicing
mRNA
U1RNP U5RNP
U4/U6RNP
splicing
The genetic information of DNA is transcribed to precursor mRNA (heterogeneous nuclear RNA, hnRNA). The small nuclear
RNAs (small nuclear RNA, snRNA) such as U1, U2, U4/U6 and U5 RNA are involved in the splicing of hnRNA. These RNAs are
complexed with proteins to form ribonucleoproteins (RNPs).
Anti-U1RNP antibody
U1 RNA
A
Sm
B
B' C
RNP
D
E
F
G
photo21
Pattern Coarse speckled (larger granular) nuclear pattern with no nucleolar and cytoplasmic staining. The
chromosomal regions do not stain in mitotic cells.
Antigen 3 kinds of polypeptides bound to U1 RNA; U1RNP-68kD, U1RNP-A(34kD), U1RNP-C(23kD)
Other analytical DID, ELISA, WB, Immunoprecipitation, CLEIA

method
Clinical The presence of the antibodies to U1RNP constitute one of major diagnostic criteria for MCTD.
ignificance
References 15, 16
14 Autoantibodies recognizing non-chromatin related proteins

Anti-Sm antibody
Pattern Coarse speckled staining similar to those of

anti-U1RNP antibody.
Antigen U1, U2, U4/U6 and U5 RNA-bound proteins

with 5 kinds of epitopes, B'/B(29kD/28kD),
D(16kD), E(12kD), F(11kD) and G(10kD).
The main epitopes comprise B'/B and D
polypeptides. Antibodies to E-F-G complex
as dominant epitopes are also reported.
Other DID, ELISA, WB, Immunoprecipitation,

analytical CLEIA
method
photo 22
AF/CDC-5* Clinical Marker antibody for SLE. The presence of
significance anti-Sm antibody is included in the criteria
for the classification of SLE by the American
College of Rheumatology.
References 17
Anti-U2RNP antibody / Anti-U1U2RNP antibody
Pattern Coarse speckled nuclear staining similar to

those of anti-U1RNP antibody.
Antigen U2RNA-bound proteins with 2 kinds of

epitopes on A'(32kD) and B"(28.5kD).
Other Immunoprecipitation
analytical
method
Clinical Polymyositis/scleroderma overlap syndrome

significance
References 3, 15, 16
photo 23

Autoantibodies recognizing non-chromatin related proteins 15

Anti-hnRNP antibody
Pattern Large speckled (granular) nucleoplasmic

staining with no chromosomal fluorescence in
mitotic cells. The antibodies frequently coexist
with anti-U1RNP and anti-Sm antibodies, and
give coarse speckled patterns.
Antigen hnRNA bound proteins-A/B, A1, A2, C, I
Other analytical ELISA, WB

method
Clinical These antibodies were first reported in

significance patients with MCTD. Antibodies to the A/
photo 24 B polypeptides and those to the A1 and A2
polypeptides occur in SLE and rheumatoid
arthritis, respectively. Recently, anti-
hnRNP-I antibodies with weak nuclear
staining specific for scleroderma and anti-
hnRNP-C antibodies with speckled nuclear
staining in scleroderma were reported.
References 18, 19, 20, 21
Anti-SS-A/Ro antibody
Y-RNA
Ro 60 kD
SS-B/La
La
Ro 52 kD
SS-A/Ro
photo 2　　　　　　　　　　　　　　　　　　　F/CDC-7*
Pattern Fine speckled nuclear staining observed in acetone-fixed slides, but not in alcohol-fixed ones.
The chromosomal region shows no fluorescence in mitotic cells.
Antigen Y1-Y5RNA-bound proteins (52kD, 60kD)
Other analytical method DID, ELISA, WB, Immunoprecipitation, CLEIA
Clinical significance These antibodies occur most frequently in patients with SS, and those with SLE. Sometimes
found in patients with rheumatoid arthritis, myositis or scleroderma. The presence of the
antibodies in the mother is associated with neonatal lupus and congenital heart block. The
presence of anti-SS-A antibodies constitutes one of the diagnostic criterion of SS in Europe.
References 22

Anti-SS-B/La antibody
Pattern Fine dense speckled nuclear staining

is observed in interphase cells with no
chromosomal fluorescence in mitotic cells.
Antigen 48 kD SS-B/La protein, a termination factor

for RNA polymerase III transcription
Other DID, ELISA, WB, Immunoprecipitation,

analytical CLEIA
method
Clinical The antibodies are found in patients with SS

significance and SLE. As with anti-SS-A antibodies, the
photo 26 presence of these antibodies is associated
AF/CDC-2* with neonatal lupus and congenital heart

block. The antibodies were reported to be
frequently positive in patients with recurrent
annular erythema. The presence of anti-
SS-B antibodies constitutes one of the
diagnostic criterion of SS in Europe.
References 23
Anti-p80 coilin antibody
Pattern Granular or few nuclear dots staining**, 0 to 6

dots (ave. 2 dots) per nucleus fluorescence.
The distinct large dotty fluorescence can be
seen in cells during S and G2 phases, and
mitotic cells show no staining.
Antigen p80 coilin, the 80kD protein composing the

coiled body in nucleoplasm.
Other WB
analytical
method
photo 27 Clinical These antibodies occur in some patients

significance with SS and those with primary biliary
cirrhosis.
References 24, 25

Autoantibodies recognizing non-chromatin related proteins 17

Anti-Sp100 antibodies
Pattern Granular or multiple (1 to 24) nuclear dots

staining**.
Antigen Sp100 protein. Multiple nuclear dots

staining is also observed by the antibodies
to promyelocytic leukemia (PML) protein,
NDP53 (a binding protein to PML oncogenic
domain including Sp100).
Other analytical WB
method
Clinical These antibodies occur in some patients

photo 28 significance with PBC.

** Since the numbers of staining dots seen in few nuclear dots and multiple nuclear dots patterns overlap each other, it is sometimes difficult to
distinguish these patterns. These patterns are often called granular or nuclear dots patterns.

■ Autoantibodies recognizing nucleolus related proteins
cytoplasm
Nucleolus
Fibrillar center:
nucleolus nuclear membrane
Nucleolus organizing regions
(N OR ) a re t h e ma in sit e s of
nucleoli formation after cell
chromatin including
division. The precursor rRNA is
transcribed from rRNA gene by
nucleus rRNA gene
RNA polymerase I.
antigen: RNA-polymerase I ~ III,
NOR-90
endoplasmic reticulum
Fibrillar component:
The processing of pre-rRNA is
performed.
precursor rRNA
antigen: fibrillarin
ribonucleoprotein
particle
Granular component:
Maturation of the rRNA is carried
mRNA out and transported outside of
the nucleolus.
ribosome antigen: PM-Scl, B23, NO55
The nucleolus is composed of the central fibrillar region and the outer granular component. In the fibrillar center, nucleolus
organizer region (NOR) is related to the reformation of the nucleolus after cell divison.
Anti-U3RNP antibody (Anti-fibrillarin antibody)
Pattern Clumpy nucleolar staining in interphase

cells with chromosomal region fluorescence
of mitotic cells.
Antigen U3RNA-bound protein; fibrillarin (34kD)
analytical
method
Clinical These antibodies occur in SSc (mainly

significance observed in patients with diffuse cutaneous
scleroderma not associated with arthritis
photo 29 and lung fibrosis), and in hepatocellular

carcinoma.
References 29, 30
Autoantibodies recognizing nucleolus related proteins 19

Anti-RNA polymerase I antibody
Pattern Punctate or speckled nucleolar staining in

interphase cells. The chromosomal regions
show no staining in mitotic cells.
Antigen RNA polymerase I, II and III component

proteins (more than 10 proteins ranged from
12.5 to 210kD). The autoantibodies targeting
RNA polymerase II or III, and those reacting to
more than one RNA polymerase were reported.
Other analytical Immunoprecipitation

method
photo 30 Clinical These antibodies occur in patients with SSc,

significance frequently associated with patients with
severe internal organ involvement (mainly
lung and kidney) and poor prognosis.
Anti-7-2RNP antibody (Anti-Th antibody)
Pattern Dotty nucleolar staining
Antigen RNaseP(8-2RNP), RNaseMRP(7-2RNP)

method
Clinical These antibodies occur in patients with

significance scleroderma, mainly associated with limited
cutaneous involvement.
References 29
photo 31
Anti-PM-Scl antibody
Pattern Homogeneous or diffuse nucleolar staining

with no chromosomal staining in mitotic cells.
Antigen The protein complex of more than 10

nucleolar proteins including 75kD and
100kD polypeptides.

significance polymyositis/scleroderma overlap syndrome
in Europe and America, but not in Japan.
References 29
photo 32
20 Autoantibodies recognizing nucleolus related proteins
Anti-NOR-90 antibody
Pattern Coarse speckled or granular nucleolar

staining in interphase cells. Several
intensely stained dots can be seen in the
chromosomal regions in mitotic cells.
Antigen 90kD/92kD proteins in nucleolus organizer

region, recognizing RNA polymerase I
transcription factor, hUBF (human upstream
binding factor) in the fibrillar center.
Other analytical DID, ELISA, WB, Immunoprecipitation

method
photo 33 Clinical These antibodies occur in patients with

significance SSc frequently associated with Raynaud's
phenomenon, also those with hepatocellular
carcinoma and SS in Japan.
References 30, 31
Anti-RNA helicase antibody
Pattern Dense speckled nucleolar staining with no

Antigen 100 kD nucleolar RNA helicase protein

method
Clinical These antibodies occur in patients with SSc,

significance SLE or watermelon stomach disease.
References 32, 33
photo 34
Other autoantibodies to nucleolar antigens:

Anti-B23/nucleophosmin antibodies occur in patients with hepatocellular carcinoma and show homogeneous nucleolar staining.
[References: 30, 34]
Anti-No55 antibodies occur in patients with interstitial cystitis and show homogeneous nucleolar pattern. [Reference: 35]
Autoantibodies recognizing nucleolus related proteins 21

■ Autoantibodies recognizing cell cycle related antigens

The remarkable differences in the patterns and intensities of nuclear staining among HEp-2 cells on the slide indicate the
presence of autoantibodies targeting the antigens which show changes in their quantities, localizations and molecular structures
dependent on the cell cycle. Sometimes, coexistence of more than two autoantibodies is suggested by the pleomorphic staining
patterns. However the details are not clear.
Anti-PCNA antibody
photo 35
PCNA: Proliferating Cell Nuclear Antigen
Pattern Most intense nuclear staining occur in S phase cells. The antibodies are characterized by the variation
of staining patterns in the cells in different cell cycles; sparse fine speckled nuclear staining in late
G1 phase cells, dense homogeneous staining in S phase cells, and coarse nuclear staining with no
nucleolar decoration in late S phase cells. The chromosomal regions show no staining in mitotic cells.
When the antibodies coexist with other antibodies, it is sometimes useful to confirm the specific staining
by serial serum dilutions.
Antigen 34kD auxiliary protein of DNA polymerase δ. Recently, it was reported that PCNA antigen forms a
macromolecule complex with Ki antigen.
Other analytical DID, WB

method
Clinical These antibodies occur in patients with SLE (1-2%), but rarely seen in other diseases.
significance
22 Autoantibodies recognizing cell cycle related antigens

Anti-Na antibody
photo 36 photo 37
Pattern Nuclear speckled staining is seen mainly in G2 phase cells, but cell cycle-dependent polymorphic
staining is not remarkable compared with that of anti-PCNA antibody. The chromosomal regions of
mitotic cells show speckled staining similar to those given by anti-centromere antibody. The cleavage
furrow observed in the telophase of mitosis and the midbody after cell division is stained by the
antibodies.
Antigen Myosin related antigen
Clinical The presence of this antibodies was reported in patients with SLE.
significance
References 39, 40
Anti-CENP-F antibody was reported to give similar staining patterns to anti-Na antibody. They occur in malignant thoracic tumors
such as lung cancer and recognize one of the centromere proteins (CENP-F 367kD). [References:44, 73]
Anti-midbody (MSA-2) antibody occuring in scleroderma show negative or very weak nuclear staining in interphase cells
compared with anti-Na antibody. [References: 43, 44]
Anti-MSA-3 antibody shows speckled staining in some of the interphase cells, but do not stain the cleavage furrow in telophase
cells. The antibodies were reported to occur in association with cancer of the respiratory system. [Reference: 44]
It was also reported that anti-DNA topoisomerase II antibodies are observed in hepatocellular carcinoma. [Reference: 30]
Autoantibodies recognizing cell cycle related antigens 23

staining patterns of the antibodies recognizing cell cycle related antigens
The photos shown below are staining patterns of the antibodies recognizing cell cycle related antigens, observed in MBL Co.,
Ltd.. Details of the antigens and the clinical significance are not determined.
photo 38 photo 39
Homogeneous nuclear staining in some of the interphase cells Weak nuclear fluorescence in some of the cells with
with more intensive staining of chromosomal region in mitotic cytoplasmic staining.
cells.
photo 40 photo 41
Some of the nucleoli are stained. No nuclear staining in interphase cells with intense
Many proteins have been found to be associated with proliferation of the cells. Among them are some of the antinuclear
antibodies with cell cycle dependency. The autoantibodies observed in patient sera may provide useful tools for studying the
structure and function of these autoantigens. [Reference: 45]
24 Autoantibodies recognizing cell cycle related antigens

■ Autoantibodies recognizing nuclear membrane related antigens
cytoplasm ribosome
pore membrane
endoplasmic reticulum
outer membrane
nuclear membrane
gp210 inner membrane
nuclear lamina lamin A

LAP1 LAP2 lamin B
lamin C
nucleus
Nuclear membrane is a lipid bi-layer, and its inner membrane is connected with nuclear lamina. Ribosomes and endoplasmic
reticulum are bound to the outer membrane. The nuclear membrane is perforated by multiple nuclear pores through which
transportation of molecules between the nucleus and the cytoplasm occurs.
Anti-lamin antibody
Pattern Linear fine staining of the nuclear

membrane and homogeneous nuclear
staining associated with nuclear rim
staining are observed. The staining is
similar to peripheral pattern. However, the
chromosomal region show no staining in
mitotic cells.
Antigen Nuclear lamins (intermediate filaments

lining the inner nuclear membrane; lamin B
(68kD), lamin A (74kD), and lamin C (60kD))
Lamina associated proteins (LAP 1A and
photo 42
LAP 2) also give continuous rim staining of
the nuclear envelope.

significance PBC, AIH, chronic fatigue syndrome and
viral hepatitis type D.
References 46, 47, 48, 49, 50
Autoantibodies recognizing nuclear membrane related antigens 25

Anti-nuclear pore complex antibody
photo 43
Pattern Nuclear membrane (pores) staining, coarse nuclear staining associated with nuclear rim staining in
interphase cells.
Antigen gp210 (210kD glycoprotein organizing nuclear pores). The antigenic epitope of this protein is located in
its cytoplasmic tail, and also epitopes in its carbohydrate moiety or amino terminal domain directed to
the lumen of the pore were reported.
Clinical PBC
significance
26 Autoantibodies recognizing nuclear membrane related antigens
Fluorescence patterns of cytoplasmic autoantigens
There are many important organelles in the cytoplasm which fulfill various functions of the cell as described in section 1. Various
autoantibodies, known as anti-cytoplasmic antibodies, targeting the proteins organizing these organelles and many cytosol
proteins have been found.
■ Autoantibodies recognizing antigens of cytoplasmic organelles

Anti-mitochondrial antibody
photo 44 photo 45
Staining of AMA-M2 using rodent kidney and stomach sections
as substrates. Cytoplasmic staining in the renal tubular
cells (intense staining in the distal tubular cells) and intense
fluoresence in the parietal cells of the stomach are observed.
Pattern Coarse granular filamentous staining extending throughout the cytoplasm.
Antigen Anti-mitochondrial antibodies-M2 (AMA-M2): pyruvate dehydrogenase (PDH-E2), located on the inner
mitochondrial membrane. Antibodies to AMA-M1 and sub-patterns of AMA-M3 to M9 were reported. The
staining shown here is due to AMA-M2 antibodies.
Other analytical IF (rodent kidney or stomach sections used as substrates), ELISA, WB, CLEIA
method
Clinical These antibodies occur frequently in patients with PBC and their presence constitutes one of the
significance diagnostic criteria of the disease. Recently, the presence of mitochondrial autoantigen in bile was
reported.
References 53, 54
Autoantibodies recognizing antigens of cytoplasmic organelles 27
Anti-ribosomal antibody
photo 46 photo 47
Staining of anti-ribosomal antibody using rodent kidney and
stomach sections as substrates. The diffuse staining in the
kidney tubular cells and fluorescence of the chief cells of the
stomach are observed. (Comparatively, anti-mitochondrial
antibodies fluoresce in the pariental cells of the stomach.)
Pattern Homogeneous cytoplasmic staining with perinuclear accentuation is associated with nucleolar staining.
Antigen 3 phosphoproteins of 60S ribosomal particles (P0(38kD), P1(19kD), and P2(17kD)), 28S ribosomal
RNA, L12 protein
Other analytical IF, DID, WB, Immunoprecipitation

method
Clinical These antibodies occur in patients with SLE associated with neuropsychiatric symptoms. Presence in
significance scleroderma patients was also reported.
28 Autoantibodies recognizing antigens of cytoplasmic organelles
Anti-Jo-1 antibody
photo 48
AF/CDC-10*
Pattern Fine granular speckled cytoplasmic staining, but weak fluorescence is usual. The granules are
condensed around the nucleus and diminish toward the periphery of the cell. The reference serum of
AF/CDC-10 from CDC rarely stains in the usual dilution of the test. It was reported that Jo-1 antigens
exist in both nucleus and cytoplasm.
Antigen Histidyl-tRNA-synthetase (catalyzing the binding reaction of histidine to 3'-OH of tRNAHis)
Other analytical DID, ELISA, Immunoprecipitation, CLEIA

method
Clinical These antibodies occur specifically in patients with PM/DM, and is frequently associated with interstitial
significance pulmonary fibrosis and polyarthritis. The presence of these antibodies consititutes one of diagnostic
criteria for PM/DM.
References 2, 58
Antibodies to other aminoacyl-tRNA-synthetases (described below) showing similar staining patterns to anti-Jo-1 antibody are
found in patients with polymyositis and dermatomyositis. [Reference: 59,74, 75]
Anti-PL-7 antibody … threonyl-tRNA synthetase
Anti-PL-12 antibody … alanyl-tRNA synthetase
Anti-OJ antibody … isoleucyl-tRNA synthetase
Anti-EJ antibody … glycyl-tRNA synthetase
Anti-KS antibody…asparaginyl-tRNA synthetase
Antibodies to the signal recognition particle (SRP) show fine dense speckled cytoplasmic staining, and is highly specific to
polymyositis. The targeting 54kD protein of SRP bind to the rough endoplasmic reticulum and ribosomes. In patients with these
antibodies, poor response to therapy and frequent recurrence are characteristically observed.[Reference: 60]

Anti-lysosomal antibody
Pattern Large speckles distributed throughout the

cytoplasm fluorescence.
Antigen Lysosomal proteins. The enzymes in

lysosomes like cathepsin and lysosome-
associated membrane proteins (LAMPs) are
considered as candidate antigens. However,
they are poorly characterized.
Clinical These antibodies occur infrequently in

significance patients with SLE. Clear clinical associations
are not known.
photo 49 References 1, 2
Anti-Golgi apparatus antibody
Pattern Irregular discontinuous granular staining in

cytoplasm around the nucleus.
Antigen 230kD protein of Golgi apparatus. Recently

97kD (golgin-97) and 200kD non-myosin
proteins were reported.
Clinical These antibodies occur infrequently in

significance patients with SLE and SS. The Golgin-97
was reported to be specific to secondary
SS.
References 61, 62
photo 50
30 Autoantibodies recognizing antigens of cytoplasmic organelles
autoantibodies recognizing the protein components of various organelles in cytoplasm
The photos shown below suggest the presence of autoantibodies recognizing the protein components of various organelles in
cytoplasm, the enzymes in vesicles and secretory granules or degraded substances taken into the vesicles. They show dense-
to-coarse cytoplasmic staining, but the corresponding antigens are not determined. Photo 54 shows the pattern relevant to cell
cycle.
photo 51 photo 52
Dense staining throughout cytoplasm. Same as photo 51, but more coarse staining.
photo 53 photo 54
Similar staining to the mitochondrial antibodies but the Some of the cell populations show cytoplasmic staining.
speckles are different.

■ Autoantibodies recognizing mitotic spindle apparatus related antigens

The main component protein of the microtubles of the spindles is tubulin, and the microtubules bind to motor proteins like kinesin
and dynein. Anti-spindle apparatus antibodies like NuMA antibodies possibly recognize the component proteins and the binding
proteins of the microtubules.
Anti-spindle apparatus antibody
Pattern The spindle fibers between the poles are

stained in mitotic cells.
Antigen Tubulin
Clinical The presence of anti-tubulin antibodies

significance were reported to occur in patients with
parasitic infections and those with Guillain-
Barre syndrome.
References 63, 64
photo 55
Anti-nuclear mitotic apparatus (NuMA)-1 antibody
Pattern The spindle poles and the spindle fibers

around them are stained in mitotic cells.
Nuclear speckled staining can be seen in
interphase cells.
Antigen 210kD protein
Other WB
analytical
methods

significance autoimmune diseases like SS.
photo 56 Reference 65
32 Autoantibodies recognizing mitotic spindle apparatus related antigens

Anti-nuclear mitotic apparatus (NuMA)-2 antibody
Pattern The spindle poles and the spindle fibers

are stained in mitotic cells. At anaphase/
telophase, the staining is hifted to the
intercellular bridge and the midbody. The
nucleus and cytoplasm show no staining in
the interphase cells.
Antigen kinesin-like protein HsEg5(130kD)
analytical
methods
photo 57 Clinical These antibodies occur in patients with

significance autoimmune diseases like SLE.
References 65, 66
Anti-centriole antibody
Pattern The poles of the mitotic spindle are stained

in the mitotic cells and one or two dots are
stained in the cytoplasm of the interphase
cells.
Antigen 48kD heat shock proteins (enolase)
Clinical The antibodies occur infrequently in patients

significance with Raynaud's syndrome and SSc.
References 67, 68
photo 58
Autoantibodies recognizing mitotic spindle apparatus related antigens 33

■ Autoantibodies recognizing cytoskeleton related antigens

Cytoskeletons are comprised of three kinds of fibrous proteins; microfilaments (5 to 6 nm in diameter), intermediate filaments (10
nm) and microtubules (25 nm). The microfilaments consist of actin and various binding proteins. The intermediate filaments in
HEp-2 cells comprise cytokeratin and vimentin. The microtubules consist of tubulins and organize the cell division apparatus.
Anti-actin antibody
photo 59 photo 60
Staining using rodent kidney and stomach sections as
substrates.
The vascular walls and the basement membrane of the
glomeruli are stained and the muscular layer and muscularis
mucosae of the stomach are stained.
Pattern Microfilaments are stained throughout the cytoplasm.
Antigen Actin
Other analytical IF, ELISA

methods
C l i n i c a l These antibodies occur in patients with AIH.

significance
References 69, 70
The autoantibodies to actin binding proteins, tropomyosin and vinculin give similar fluorescence to those given by anti-actin
antibodies. [References 1]
34 Autoantibodies recognizing cytoskeleton related antigens

Anti-cytokeratin antibody
Pattern Reticular cytoplasmic staining (weak to

intense fluorescence depending on the
cells) and granular staining in mitotic cells
are seen.
Antigen Cytokeratin (40 to 52kD intermediate

filament)
Other WB
analytical
methods
Clinical These antibodies are frequently observed

significance in patients with RA and also occur in other
photo 61
diseases. The presence of antibodies in
early RA patients with negative rheumatoid
factor was reported.
References 71
Anti-vimentin antibody
photo 62 photo 63
Staining using rodent kidney and stomach sections as
substrates.
The vascular wall of kidney and stomach mucous membrane
are stained, but it is different from the staining produced by
anti-actin antibodies.
Pattern Cobweb-like cytoplasmic staining with denser perinuclear staining.
Antigen Vimentin (53kD intermediate filament)
Other analytical WB
methods
Clinical The antibodies occur in patients with RA, liver diseases and chronic fatigue syndrome.
significance
References 48, 72
Autoantibodies recognizing cytoskeleton related antigens 35

Other fluorescence patterns
■ Discussion about staining of “rods and rings (RR)” in Fluoro HEPANA test (HEPANA)
In our sample staining service for the customers using

HEPANA, there are specific patterns which look like “rod-type”
and “ring-type “fluorescent staining in a cytoplasm. These
are called “cytoplasmic rods and rings (RR)” as reported by
several papers. In recent knowledge, it became clear that
RR is detected specifically in serum of patients with chronic
hepatitis C. Among them, RR is detected more frequently in
hepatitis C patients receiving combination therapy of pegylated
Interferon (PEG-IFN) and ribavirin (RBV). It also seems that
so far, there are no reports that RR was detected in patients
without any treatment. The current study reports CTPS1 and
IMPDH2 were identified as components associated with RR.
CTPS1 and IMPDH2 play role in the biosynthetic pathway for CTP and GTP, respectively, which are as nucleotide sources for
DNA/RNA. It is known that RR appears when the CTP or GTP synthetic pathway is inhibited, and it is also reported that RR
formation was observed even when HEp-2 cells themselves were treated with CTPS1 or IMPDH2 inhibitor. The ribavirin is an
IMPDH2 inhibitor, so that RR may be formed in hepatitis C patients who were actually treated with ribavirin. If the formed RR
was released somehow into the blood, the immune system would recognize it as a foreign substance to induce autoantibodies
to RR. This is the assumed mechanism of RR staining detected by sera from patients with hepatitis C. As possible mechanisms
for formation of RR in HEp-2 cells, it is reported that RR was formed by not only inhibition of CTP and GTP synthetic pathway,
but also other conditions, such as nutrient starvation (lack of glucose), addition of sodium azide, and treatment with some kinds
of kinase inhibitor. In case of our HEPANA, MBL has experience that the frequency of RR formation is dependent on the lot of
the calf serum used for HEp-2 cell culture. Such lot-to-lot variation may provide different nutritional conditions to form RR. In
fact, HEp-2 growth rate seems to be variable according to the lot of calf serum. Together with, we assume that when HEp-2
cells are exposed with a certain lot of calf serum which tend to provide a starvation, RR formation would be induced in the cells.
[reference76-80]
36 Other fluorescence patterns

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40 Reference

Atlas of Antinuclear Antibodies

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Atlas of Antinuclear Antibodies

Structure and function of eukaryotic cell and outlines of cell cycle

■ Structure and function of cell endoplasmic reticulum nucleus nuclear membrane

membrane and organelles are occupied by insoluble

Fig. 1 HEp-2 cell

Endoplasmic reticulum (ER)

Table 1 Antigens recognized by autoantibodies in connective tissue diseases

antinuclear antibodies localization recognized antigens relevant diseases

anti-ssDNA antibody chromatins single stranded DNA (ssDNA) SLE etc.

anti-U1RNP antibody nucleus U1RNP MCTD

anti-U3RNP antibody nucleolus U3RNP (Fibrillarin) SSc

anticytoplasmic antibodies localization recognized antigens relevant diseases

anti-Jo-1 antibody cytoplasm histidyl-tRNA synthetase PM/DM

*Staining patterns of anti-centromere antibody in photo.

centriole connected by the polar microtubules.

microtubule arrangement is reformed,

After mitosis, cytokinesis occurs by the formation of

Fluorescence patterns of autoantibodies

■ Typical patterns of ANA using HEp-2 cells

No significant nuclear and cytoplasmic staining. Depending on the

In case of positive, staining patterns are interpreted.

Homogeneous nuclear staining

Uniform diffuse fluorescence staining of the entire nucleus in

Fluorescence patterns of autoantibodies 5

Peripheral nuclear staining

Uniform diffuse nuclear staining similar to homogeneous staining,

Speckled nuclear staining

Fine or coarse speckles are distributed in the entire nucleus of

Centromere (Discrete speckled) staining

40 to 80 discrete speckles are stained in the nucleus of interphase

6 Fluorescence patterns of autoantibodies

Homogeneous or speckled (clumpy) staining of the nucleolus of

Other staining patterns and antigens,

Cell cycle-dependent pleomorphic proliferating cell nuclear antigen (PCNA)

Nuclear membranous staining lamin, nuclear pore complex

Cytoplasmic staining cytoplasmic antigens; enzymes such as aminoacyl-tRNA synthetases, organelles

Fluorescence patterns of autoantibodies 7

show another example of the mixed pattern

Fluorescence patterns of nuclear autoantigens

■ Autoantibodies recognizing chromatin related antigens

chromosome "beads-on-a-string" form of chromatin

photo13 photo14 AF/CDC-1*

10 Autoantibodies recognizing chromatin related antigens

Antigen Double stranded DNA(dsDNA). Anti-ssDNA antibodies do not give fluorescence.

Other analytical IIF, ELISA, RIA, CLEIA

Anti-DNA histone complex antibody (anti-DNP antibody / anti-nucleosomal antibody)

Pattern Homogeneous nuclear staining in

Antigen Histone-DNA complex with the antigenic

Clinical SLE, lupus nephritis

* Reference serum from Centers for Disease Control

Autoantibodies recognizing chromatin related antigens 11

Pattern Similar to the former antibody

Other analytical ELISA, WB

Pattern Speckled (patchy) nuclear staining (sometimes

Antigen DNA binding 80kD/70kD heterodimer protein

Other DID, Immunoprecipitation

Clinical SLE, SSc (USA) / polymyositis/scleroderma

Anti-Scl-70 antibody (anti-Topoisomerase I antibody)

Pattern Homogeneous (dense fine speckled to

Antigen DNA Topoisomerase I

Other DID, ELISA, WB, CLEIA

Clinical Antibodies to topoisomerase I are generally

photo 18 sensitivity and their presence constitutes one

AF/CDC-9* of the diagnostic criteria of the disease.

photo13 photo14 　　AF/CDC-1*